8.5:
Proofreading
During DNA replication, complementary base pairing dictates the sequence of nucleotides added to the new strand — adenine pairs with thymine and guanine pairs with cytosine.
However, nucleotides can sometimes be incorrectly paired, for instance, adenine with cytosine. Such errors are prevented or repaired by the proofreading properties of DNA polymerases carried out during DNA synthesis.
First, DNA polymerase has a high affinity for complementary nucleotides, which improves the accuracy of selecting the correct incoming nucleotide to pair with the template strand.
Second, as the nucleotides start pairing, the DNA polymerase undergoes a conformational change that makes the mispaired nucleotides more likely to dissociate but retains the correctly paired nucleotides.
Third, if an incorrect nucleotide is somehow added to the growing 3' end, the structural deviation caused by this incorrect pairing causes the DNA polymerase to pause.
In a process called exonucleolytic proofreading, the 3' end is then transferred to the exonuclease site of the DNA polymerase. The polymerase subsequently removes the incorrect nucleotide in a 3' to 5' direction, replaces it with the correct nucleotide, and resumes DNA synthesis.
8.5:
Proofreading
Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore, it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Genomic DNA is synthesized in the 5’ to 3’ direction. Each cell contains a number of DNA polymerases that play different roles in synthesizing and correcting mistakes in the DNA. For example, DNA polymerase delta and epsilon possess proofreading ability when replicating nuclear DNA. These polymerases “read” each base after it is added to the new strand. If the newly-added base is incorrect, the polymerase reverses direction (moving from 3’ to 5’) and uses an exonucleolytic domain to cut off the incorrect base. Subsequently, the excised base is replaced with the correct base.
Mutations in the Exonuclease Domain of DNA Polymerase are Linked to Cancers
Proofreading is important for preventing mutations from occurring in newly-synthesized DNA, but what happens when the proofreading mechanism fails? When a mutation alters the exonuclease domain of DNA polymerase, it loses the ability to remove incorrect nucleotides. In consequence, mutations can accumulate rapidly throughout the genome. This type of mutation has been linked to various types of cancer.
Low-fidelity DNA Polymerase can Generate Mutated DNA Sequences
Modified DNA polymerases are used in laboratory science for polymerase chain reaction (PCR), an in vitro technique for making many copies of specific fragments of DNA. While high-fidelity polymerases are used when it is important that the end product is perfect, some techniques, such as error-prone PCR, seek to generate mutations in a stretch of DNA on purpose. These techniques use polymerases that have compromised proofreading ability.